Field of the Invention
The present invention relates to a light sheet fluorescence microscope and a technique related to the light sheet fluorescence microscope.
Description of the Background Art
In recent years, light sheet fluorescence microscopes have been widely used, and improvements have been actively made upon the light sheet fluorescence microscopes in the life science field.
In the observation of a biological sample by a light sheet fluorescence microscope, the biological sample is labeled with a fluorescent dye, the labeled biological sample is illuminated laterally by sheet-shaped illumination light, and fluorescence emitted from the illuminated portion is received. As a result, a sectioned image of the illuminated portion is achieved at high speed.
The technique of laterally illuminating a sample, however, has points to be improved.
The first point that needs improvement is that the conventional technique fails to achieve both a wide field of view (FOV) and a high Z-direction resolution. In a typical light sheet fluorescence microscope, a laser is used as a light source, and a laser beam emitted from the laser is focused into a sheet shape, creating a light sheet. Due to the effect of diffraction, the light sheet has a finite expanse through its thickness at a focus position. The resolution of the light sheet fluorescence microscope through its thickness is determined by the beam waist size of the light sheet, and thus, is improved by reducing the beam waist of the light sheet. If the beam waist of the light sheet is reduced, however, the beam size increases abruptly as away from the focus position, resulting in a smaller FOV having a uniform resolution through the thickness of the light sheet. In the light sheet fluorescence microscope, thus, the beam waist of the light sheet is normally adjusted such that the beam expands uniformly and that the Z-direction resolution accordingly becomes uniform within a desired FOV. Thus, a typical light sheet fluorescence microscope sacrifices the Z-direction resolution.
In the light sheet fluorescence microscope, an observation objective lens is arranged for observation such that the optical axis of the observation objective lens is perpendicular to the optical axis of an illumination objective lens. The depth of field (DOF) of the observation objective lens is normally smaller than the beam waist of the light sheet adjusted to have a uniform Z-direction resolution within a desired FOV. Thus, the fluorescence emitted from outside the DOF is not imaged sufficiently and forms a blurred image that does not clearly indicate sample structure information. The florescence then becomes background light and degrades the contrast of a final image finally acquired.
Further, if a highly scattering sample is observed, not only the fluorescence emitted from outside the DOF but also scattered light becomes background light, degrading the contrast of the image finally acquired. This makes it difficult to achieve a high contrast image of a highly scattering sample.
The second point that needs improvement is shadowing. The light sheet fluorescence microscope laterally illuminates a sample, and if the sample contains a portion having a high absorption, the illumination light does not reach the back of the relevant portion, forming a shadow. The illumination light does not reach the shadow, and accordingly, a fluorescence label is not excited in the shadow, so that the shadow remains as an artifact in the final image finally acquired.
To make improvements upon the first and second points that need improvement, structured illumination and pivoting illumination have been proposed and demonstrated. U.S. Pat. No. 8,970,950 (Patent Document 1); U.S. Pat. No. 9,223,125 (Patent Document 2); Keller and five others, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy”, Nature Methods (US), 2010, Vol. 7, Issue 8, pp 637-642 (Non-Patent Document 1); Chen and 25 others, “Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution”, Science (US), 2015, Vol. 346, Issue 6208, pp 1257998-1-1257998-12 (Non-Patent Document 2); and Huisken et al., “Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM)”, Optics Letters (US), 2007, Vol. 32, Issue 17, pp 2608-2610 (Non-Patent Document 3) are examples of such illumination.
In the light sheet fluorescence microscope with structured illumination, a sample is illuminated by three or more types of light sheets that have a sinusoidal intensity distribution but respectively have three or more types of different phases. Additionally, three or more images are individually acquired during the illumination of the sample by the three or more types of light sheets. Further, the acquired three or more images are subjected to simple image processing to remove the background light that forms a blurred image, so that a final image having a high contrast is acquired. The techniques described in Non-Patent Documents 1 and 2 are examples of such techniques.
In the light sheet fluorescence microscope with pivoting illumination, a sample is sequentially illuminated by two or more light sheets having different propagation angles. Consequently, the illumination light reaches the back of the portion having a high absorption, reducing the influence of the shadow. The techniques described in Patent Document 1 and Non-Patent Document 3 are examples of such techniques. The techniques for pivoting illumination are specific to light sheet fluorescence microscopes.
The light sheet fluorescence microscope with structured illumination typified by the techniques described in Patent Document 2 and Non-Patent Document 2 uses a liquid crystal on silicon-spatial light modulator (LCOS-SLM) to create a light sheet having a sinusoidal intensity distribution. The microscope also uses galvanometer mirrors to shift the phase of the intensity distribution. The phase shift speed of the intensity distribution is thus limited by the scanning speed of the galvanometer mirrors. On the other hand, the scanning speed of the galvanometer mirrors is several tens of kHz at most. Faster image acquisition is thus difficult in the light sheet fluorescence microscope.
In digital scanned laser light sheet fluorescence microscopy with incoherent structured illumination microscopy (DSLM-SI) described in Non-Patent Document 1, a linearly focused laser beam is scanned by a galvanometer mirror in the focal plane of the observation objective lens to create a phantom light sheet. Also, to create structured illumination, the intensity of a laser beam is subjected to faster temporal modulation by an acousto-optic modulator (AOM) in synchrony with scanning of the laser beam. The modulation speed of the AOM is normally about several MHz. The frequency of the structured illumination is limited by the size of the focused beam waist even when the modulation rate of the AOM is high. The frequency of the structured illumination is an important parameter for determining sectioning capability, and the sectioning capability is improved more as the frequency of the structured illumination is higher. For this reason, the limitation of the frequency of the structured illumination by the size of the focused beam waist tends to inhibit an improvement in sectioning capability. Additionally, the system tends to be complicated because, for example, the DSLM-SI needs to bring the modulation by the AOM into synchronization with scanning by the galvanometer mirror.
The pivoting illumination typified by the pivoting illumination described in Non-Patent Document 3 uses a galvanometer mirror to switch the propagation direction of the light sheet. The speed of switch of the propagation direction is thus limited by the scanning speed of the galvanometer mirror. On the other hand, the scanning speed of the galvanometer mirror is several tens of kHz at most. Faster image acquisition is difficult in pivoting illumination.
As described above, the conventional structured illumination and pivoting illumination have a problem of the difficulty in acquiring an image at higher speed. Additionally, the conventional structured illumination has a problem of the difficulty in increasing the frequency of the structured illumination.